Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands.
J Virol Methods. 2013 Dec;194(1-2):146-53. doi: 10.1016/j.jviromet.2013.07.050. Epub 2013 Aug 18.
Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types in vitro and in tissues in animal models is important for understanding its pathogenesis. As detection of the negative strand viral RNA is a more reliable indicator of active replication for single-stranded positive-sense RNA viruses, the specificity of qRT-PCR assays currently used for the detection of WNV positive and negative strand RNA was reassessed. It was shown that self- and falsely-primed cDNA was generated during the reverse transcription step in an assay employing unmodified primers and several reverse transcriptases. As a result, a qRT-PCR assay using the thermostable rTth in combination with tagged primers was developed, which greatly improved strand specificity by circumventing the events of self- and false-priming. The reliability of the assay was then addressed in vitro using BV-2 microglia cells as well as in C57/BL6 mice. It was possible to follow the kinetics of positive and negative-strand RNA synthesis both in vitro and in vivo; however, the sensitivity of the assay will need to be optimized in order to detect and quantify negative-strand RNA synthesis in the very early stages of infection. Overall, the strand-specific qRT-PCR assay developed in this study is an effective tool to quantify WNV RNA, reassess viral replication, and study tropism of WNV in the context of WNV pathogenesis.
研究西尼罗河病毒（WNV）在体外不同细胞类型和动物模型组织中的趋向性和复制动力学对于理解其发病机制非常重要。由于检测负链病毒 RNA 是单链正链 RNA 病毒活性复制的更可靠指标，因此重新评估了目前用于检测 WNV 正链和负链 RNA 的 qRT-PCR 检测方法的特异性。结果表明，在使用未经修饰的引物和几种逆转录酶的检测中，在逆转录步骤中会产生自我和错误引发的 cDNA。因此，开发了一种使用耐热 rTth 结合标记引物的 qRT-PCR 检测方法，通过避免自我和错误引发事件，大大提高了链特异性。然后在体外使用 BV-2 小胶质细胞和 C57/BL6 小鼠评估了该检测方法的可靠性。可以在体外和体内跟踪正链和负链 RNA 合成的动力学；然而，需要优化该检测方法的灵敏度，以便在感染的早期阶段检测和定量负链 RNA 合成。总之，本研究中开发的这种链特异性 qRT-PCR 检测方法是一种有效的工具，可用于定量WNV RNA、重新评估病毒复制，并研究WNV 在 WNV 发病机制中的趋向性。
